View source for Walkthrough on command line based tilt series alignment (Tsinghua Spring School 2025)

[[Category:WalkthroughsSpecific]]

Dynamo includes a package for automated alignment and reconstruction of tilt series. This walkthrough guides users through a series of steps on how to use it in command line based mode. The [[Walkthrough on GUI based tilt series alignment | GUI based alignment walkthrough]] shows how to operate this procedure through the GUI. Here, we go through each command one by one (for advanced applications, all commands can also be summarized in a Matlab script and run at once).

= Data =

We use a binned version of the first tilt series from the EMPIAR entry 10164, depicting a set of virus like particles (VLP). You can download it directly from EMPIAR, then bin it,   <!--[https://drive.google.com/file/d/1EsnKBvbFuWD1njk8dRVLc3SlhdsTHIpY/view?usp=drive_link or simply follow this link]-->[{{Template:DownloadLinkAWFHIV1TS}} or simply follow this link] to download a 600Mb file. You can also follow this tutorial using your own data.

In this course, the data for this walkthrough is in:
 <tt > /mnt/fs/2025workshop/Users/user**/Data/DownloadLinkAWFHIV1TS/</tt>
and the tomogram with the data is in the link:
 <tt > /mnt/fs/2025workshop/Users/user**/Data/DownloadLinkAWFHIV1TS/b001ts001.mrc </tt>

= Create the workflow =
The following commands create a new alignment workflow in a separate folder. Make sure ''Dynamo'' is running and type the following commands into the Matlab command line (or the ''Dynamo'' console, if you work on the standalone version):
 
 <nowiki>name = 'hivCommandLine';
folder = 'workflows';
u = dtsa(name,'--nogui','-path',folder,'fp',1); </nowiki>

In the Matlab workspace you should now see the variables that you just created with these commands. If you work with the standalone version you can simply type the word <tt>whos</tt> to see a list of all variables in the workspace.

= Entering the data =

The variable <tt>u</tt> is an object that contains several ''fields'' to interact with the workflow. They can be found through autocompletion by using the <tt>tab</tt> key (or by pressing the enter key in the standalone version). In the following, these different fields will be used to pass our inputs to the workflow.

[[ File:alignCommandLine_autocompletionOfUserObject.png |thumb|center| 700px|autocompletion of user object to access different fields in matlab and standalone version]]

== Basic data==
Basic data can be entered using the ''enter'' field. Here we use a binned version of the first tilt series from the EMPIAR entry 10164, depicting a set of virus like particles (VLP). Visit the course materials to access the tilt series or download it directly from EMPIAR (you can also use your own data). The file of the tilt series and the angles are passed to the workflow:

 <nowiki>u.enter.tiltSeries('b001ts001.mrc');
u.enter.tiltAngles(-57:3:60); </nowiki>

If necessary (not in this tutorial), you can use the ''discardedTiltIndices'' field to reject tilts that are of too bad quality using the following command (e.g., tilt 1 and 40):
 <tt>u.enter.discardedTiltIndices([1,40]); </tt>

== Acquisition settings==
Here you can enter parameters such as <tt>Cs</tt>, nominal defocus, etc. For this tutorial we just provide the pixel size in Angstroms:
 <tt>u.enter.settingAcquisition.apix(2.7);</tt>

== Computation settings==
We enable the use of parallel cores
 <tt>u.enter.settingComputing.parallelCPUUse(1); </tt>
This will be using mainly for gold bead detection (cc computation) and reconstruction.

To use all available physical cores use:
 <tt>u.enter.settingComputing.cpus('*');</tt>

== Detection settings==
These here are the actual design decisions when running an alignment workflow. The gold bead radius has to be known beforehand ( see [[Walkthrough on GUI based tilt series alignment | GUI based alignment walkthrough]] on how to measure it). Binning is used internally only for quick and robust bead detection.

 <nowiki>u.enter.settingDetection.detectionBinningFactor(2);
u.enter.settingDetection.beadRadius(16);
u.enter.settingDetection.maskRadius(28);
u.enter.templateSidelength(64);</nowiki>

== Changing generic parameters ==
You can find all parameters of the individuals steps using autocompletion on the <tt>area</tt> field and then by adding <tt>step</tt> in the end. For example, the residual threshold of the tilt gap filler can be changed as follows:
 <tt>u.area.indexing.step.tiltGapFiller.parameterSet.residualsThreshold(8);</tt>

= Running the workflow =
After setting the input parameters, the complete workflow can be run using the following command (we skip the CTF correction here). It should take about 5 minutes to run.
 <tt>u.run.all('noctf',1);</tt>

== Checking the results ==
After completion, the <tt>view</tt> field allows you to check different output items for diagnose, such as the tilt lines:

 <tt>u.view.tiltLines.unaligned();</tt>

[[ File:alignCommandLine_viewOfTiltLines.png |thumb|center| 600px|view of tilt lines]]

or the different reconstructions 

 <nowiki>u.view.reconstruction.binnedWBP();
u.view.reconstruction.binnedSIRT();</nowiki>

or the aligned stacks themselves.

 <tt>u.view.stack.alignedFull();</tt>

You can also use the autocompletion of the <tt>info</tt> field (as shown in the beginning) to find and display different logs:

 <nowiki>>> u.info.fit;
------------------------------------------------------------
File with info:
  workflows/hivCommandLine.AWF/info/fitting.doc
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 
rms:  1.41
psi: 85.20
fit modus: normal
total markers: 79
total observations: 2702
fit created at: 11-Aug-2021 10:56:24
------------------------------------------------------------</nowiki>

 <nowiki>>> u.info.markers;
------------------------------------------------------------
Info on working markers [used during detection, indexing refinement]
  ... item saved and in memory
Item in memory:
Number of shapes          :  79
Number of frames          :  40
Number of observations    :  2702 (out of 3160)
Number of empty frames    :  1
Number of complete frames :  0
Number of complete shapes :  0
 
Item in disk:
Markers in disk and currently in memory contain equal coordinates
 
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 
 
Info on final markers [used from alignment step]
  ... item saved and in memory
Item in memory:
Number of shapes          :  79
Number of frames          :  40
Number of observations    :  2702 (out of 3160)
Number of empty frames    :  1
Number of complete frames :  0
Number of complete shapes :  0
 
Item in disk:
Markers in disk and currently in memory contain equal coordinates
------------------------------------------------------------</nowiki>

If you are following this tutorial during a ''Dynamo'' workshop, the exercise ends here.

= On the fly reconstruction of particles=

In this section we show how to avoid having to reconstruct a '''full''' tomogram at '''full''' resolution for the case where only a set of subtomograms are need. For this exercise we will use the gold beads as proxies that represent any particles of interest. We will first locate a few of them in the '''binned''' tomogram, and then use those coordinates to directly reconstruct the subtomograms in full resolution and skipping the need to reconstruct the whole full sized tomogram..

== Define coordinates in the binned tomogram==

First, we open the binned reconstruction tht we created before using the visualization tool <tt>dtmslice</tt> and adjust the contrast if necessary (arrow):

 <tt>dtmslice 'workflows/hivCommandLine.AWF/reconstruction/binnedReconstructionWBP.mrc';</tt>

[[ File:alignCommandLine_dtmsliceOfBinnedReconstruction.png |thumb|center| 600px|dtmslice of binned reconstruction]]
[[ File:alignCommandLine_dtmsliceAfterContrastCorrection.png |thumb|center| 600px|dtmslice after contrast correction]]
To manually define the particle coordinates, we first open a new model of type <tt>general</tt>:
[[ File:alignCommandLine_createANewModel.png |thumb|center| 600px|create a new model]]
To quickly find the 3d positions of a set of particles, we use a projection of the tomogram:
[[ File:alignCommandLine_showAFullProjection.png |thumb|center| 600px|show a full projection]]
[[ File:alignCommandLine_fullProjectionOfBinnedReconstruction.png |thumb|center| 600px|full projection of binned reconstruction]]
Right click on the (x,y) position of a particle opens two small windows showing the x-z and y-z planes:
[[ File:alignCommandLine_rightClickOnAPoint.png |thumb|center| 600px|right click on a point]]
Use the y-z depiction to left clicking on the marker. After clicking, the marker coordinates are saved in the model:
[[ File:alignCommandLine_xViewOfClickedPoint.png |thumb|center| 600px|x view of clicked point]]
[[ File:alignCommandLine_mainClickToFixThe3dCoordinates.png |thumb|center| 600px|main click to fix the 3d coordinates]]
Go back to the projection view and repeat the procedure until you have a few more markers (but no more than ten in this exercise). The x-z and y-z planes will be updated automatically when clicking on new markers on the projection and do not need to be closed every time. When you are done, you can close all the plane views and also the projection view.
[[ File:alignCommandLine_clickAroundTenMarkers.png |thumb|center| 600px|click around ten markers]]
To save the coordinates we first export them into the Matlab workspace in table format:
[[ File:alignCommandLine_exportATableIntoTheMatlabWorkspace.png |thumb|center| 600px|export a table into the matlab workspace]]
The Matlab shell should report the variable name used to store the table:
[[ File:alignCommandLine_theCommandLineShowsTheVariableNameAssignedToTheCreatedTable.png |thumb|center| 400px|the command line shows the variable name assigned to the created table]]

Now we can save the table with the coordinates as a file in the current directory (this table will be used to crop the subtomograms in the next steps):
 <tt>dwrite(temp_table,'binnedGoldbeads.tbl');</tt>

=== Cropping the gold bead particles ===

We can now crop the particles using the table file that we generated before:
 <nowiki>tomo = 'workflows/hivCommandLine.AWF/reconstruction/binnedReconstructionWBP.mrc';
tbl  = 'binnedGoldbeads.tbl';
targetBinnedParticles = 'binnedGoldBeads.Data';
sidelengthBinned = 32;
o = dtcrop(tomo,tbl, targetBinnedParticles, sidelengthBinned);</nowiki>

To monitor if everything worked correctly, we visualize a projection along <tt>z</tt> of each cropped particle using the command:
 <tt>dslices(targetBinnedParticles,'proj','c');shg;</tt>

[[ File:alignCommandLine_dslicesOnBinnedGoldBeads.png |thumb|center| 600px|dslices on binned gold beads]]
== Reconstruct particles on the fly ==

The command that reconstructs full sized particles from an aligned stack is <tt>dynamo_table_trec</tt>. This is an independent function, not part of the workflow, implying that we will need to perform some format conversion.

 <tt>tbl     = dread('binnedGoldbeads.tbl');</tt>

We scale the table. "Bin level 2" means that each pixel in the binned tomogram is 2 times twice its size in the full resolution tomogram. This is the factor that we input into <tt>dynamo_table_rescale</tt>:
 <nowiki>alignmentBinLevel = 2;
tblFull = dynamo_table_rescale(tbl,'factor',2^alignmentBinLevel); </nowiki>

Now we fetch the full sized aligned stack stored inside the alignment folder:
 <tt>source  = 'workflows/hivCommandLine.AWF/align/alignedFullStack.mrc';</tt>

We create a data folder to contain the particles to be reconstructed on the fly:
 <tt>targetDirectory = 'tempCrop.Data'</tt>;

Angles ''actually'' used during the reconstruction are contained in the ''reconstructionTiltAngles' item of the output folder. Note that they might differ from the nominal angles, as some views might have been eliminated, both explicitely by the user, of by the program not being able to align all tilts.
 <tt>angles =  'workflows/hivCommandLine.AWF/align/reconstructionTiltAngles.tlt';</tt>

The (binned) coordinates were measured in a binned tomogram, whose dimensionality is requested by <tt>dynamo_table_rec</tt>. We need to provide <tt>dynamo_table_rec</tt> with 
 <nowiki>visualizationTomogram =  'workflows/hivCommandLine.AWF/reconstruction/binnedReconstructionWBP.mrc';
sizeTomogram = dynamo_read_size(visualizationTomogram);
sizeTomogramFull = sizeTomogram*2^alignmentBinLevel;</nowiki>

The tomogram center can be shifted in order to account for a possible shift in the centre in the binned reconstruction that was used to crop the particles.
 <tt>shiftTomogramCenter = [0,0,0];</tt>
We proceed now to the reconstruction. Our sid length should be 4 times the size we used before:
 <tt>sidelengthFullSize = 128;</tt>
We use the <tt>applyRampFilter</tt> option in order to explicitely estate the kind of filter that we want during the back projection.
 <nowiki>dynamo_table_rec(tblFull,source,angles,targetDirectory, sidelengthFullSize, 'applyRampFilter',1, 'sizeTomogram',sizeTomogramFull, 'shiftTomogramCenter',shiftTomogramCenter);</nowiki>

We can now check that the particles have been reconstructed in full size:
 <tt>figure;dslices(targetDirectory,'proj','c');shg;</tt>
[[ File:alignCommandLine_dslicesOnFullSizedGoldBeads.png |thumb|center| 600px|dslices on full sized gold beads]]